Epigenetic control of gene expression is employed in animals and plants to enable genetically identical cells to pursue different fates, and such cell differentiation is central to their development. Epigenetic modification and information transfer may also be employed during the bacterial infection cycle so as to change the expression of virulence functions temporally and spatially within the host. These alterations in gene expression may accompany bacterial dissemination among host sites, and in response to host inflammation, tissue breakdown, and immune reactions. In Aim 1, we propose to test the hypothesis that a methylation-based epigenetic system controls Salmonella pathogenesis. Our proposal is based on altering the expression of the bacterial methyltransferase, termed DNA adenine methylase (Dam), which is involved in the formation of DNA methylation patterns and is known to be required for virulence in a number of pathogens. We propose to determine whether transient overexpression of Dam activity in S. typhimurium leads to the establishment of a lineage of genotypically wild-type, epigenetically modified cells that exhibit altered DNA methylation patterns and altered virulence states that persist for a number of cell generations. If so, the present state of virulence gene expression in a given bacterial cell may be dependent on its past history (i.e., cellular memory), similar to that observed in cell-differentiation and developmental programs in higher organisms. In Aim 2, we propose to specify the mechanism by which cellular memory control occurs at the level of a single gene using the S. typhimurium pef operon as a model system. Plasmid encoded fimbriae (Pef) expressed by S. typhimurium mediate adherence to intestinal epithelium and are required for virulence. The pef operon is under methylation-dependent transcriptional regulation similar to the well characterized E. coli pap operon, which encodes important virulence determinants in urinary tract infections. We propose to test whether Dam overproduction-mediated defects in pef expression are a direct consequence of altered DNA methylation patterns that map to specific upstream pef Dam-target sites (GATC sequences). We further propose to determine whether transient exposure to dam overexpression leads to persistent changes in Pef synthesis and whether such changes are heritably maintained via altered DNA methylation patterns at upstream Per GATC sites. The finding of a methylation-based memory system in bacteria would provide profound insights into the fundamental molecular mechanism(s) underlying control of virulence gene expression and the resultant changes in pathogen behavior that are critical to infection. Such information is vital toward developing novel antimicrobials and vaccines against biowarfare agents and emerging infectious diseases that currently threaten public health worldwide.